Agents for use in the therapeutic or prophylactic treatment of myopia or hyperopia

ABSTRACT

The present invention relates to agents for use in the prophylactic or therapeutic treatment of myopia in a subject, wherein said agents are capable of decreasing epidermal growth factor receptor (EGFR) signaling and/or signaling of another receptor susceptible for amphiregulin in a subject in a direct or indirect manner. The present invention further relates to agents for use in the prophylactic or therapeutic treatment of hyperopia in a subject, wherein said agents are capable of increasing epidermal growth factor receptor (EGFR) signaling and/or signaling of another receptor susceptible for amphiregulin in a subject. Furthermore, the present invention relates to methods for the diagnosis of myopia or hyperopia in a subject, comprising the steps of (a) providing a biological sample from the subject; (b) determining the amphiregulin level in said sample; (c) comparing the level determined in step (b) to the amphiregulin levels found in emmetropic jects or subjects going to be emmetropic; and (d) determining that the subject has myopia or is predisposed for the development of myopia in case the level determined in step (b) is higher than the amphiregulin levels found in emmetropic subjects or subjects going to be emmetropic; and determining that the subject has hyperopia or is predisposed for the development of hyperopia in case the level determined in step (b) is lower than the amphiregulin levels found in emmetropic subjects or subjects going to be emmetropic. Finally, the present invention relates to a method for identifying agents which associate with amphiregulin or fragments or variants thereof.

The present invention relates to agents for use in the prophylactic ortherapeutic treatment of myopia in a subject, wherein said agents arecapable of decreasing epidermal growth factor receptor (EGFR) signalingand/or signaling of another receptor susceptible for amphiregulin in asubject in a direct or indirect manner. The present invention furtherrelates to agents for use in the prophylactic or therapeutic treatmentof hyperopia in a subject, wherein said agents are capable of increasingepidermal growth factor receptor (EGFR) signaling and/or signaling ofanother receptor susceptible for amphiregulin in a subject. Furthermore,the present invention relates to methods for the diagnosis of myopia orhyperopia in a subject, comprising the steps of (a) providing abiological sample from the subject; (b) determining the amphiregulinlevel in said sample; (c) comparing the level determined in step (b) tothe amphiregulin levels found in emmetropic subjects or subjects goingto be emmetropic; and (d) determining that the subject has myopia or ispredisposed for the development of myopia in case the level determinedin step (b) is higher than the amphiregulin levels found in emmetropicsubjects or subjects going to be emmetropic; and determining that thesubject has hyperopia or is predisposed for the development of hyperopiain case the level determined in step (b) is lower than the amphiregulinlevels found in emmetropic subjects or subjects going to be emmetropic.Finally, the present invention relates to a method for identifyingagents which associate with amphiregulin or fragments or variantsthereof.

Myopia has rapidly emerged as a global health concern in the last threedecades. It is one of the leading causes of visual impairment, and- isassociated with potentially blinding ocular complications includingretinal detachment, myopic maculopathy, glaucoma and cataract. Evidencefrom family and twin studies supports the notion that myopia can behighly heritable in some families. On the other hand, the rapid upsurgeof myopia in the last few decades in many parts of the world is likelyto be a consequence of changes in lifestyle, such as the increasingintensity of education, particularly in urban East Asia. Parallel to theincrease in the prevalence of myopia, the prevalence of hyperopia hasdeclined. However, hyperopia has marked disadvantages such as the needfor glasses for the distance and the near, and an increased prevalenceof angle-closure glaucoma, age-related macular degeneration and diabeticretinopathy.

The process of emmetropization (i.e., sharp distant vision withoutglasses and without accommodative effect of the intraocular naturalcrystalline lens) of the growing eye from the stage of an infant towardsthe stage of an adult has not been cleared yet. The development of highaxial myopia can be considered as an uncontrolled process ofemmetropization, leading to an axial elongation of the globe. A globetoo long in relationship to its refractive power focuses the image of anobject, located in the distance, in front of the retina, so that themacula as the anatomical-optical center of the retina is not located inthe focus and thus receives a blurred image with blurred borders. Thesame applies for a hyperopic globe which is too short in relationship toits refractive power and which focuses the image of an object, locatedin the distance, behind the retina.

At birth, the axial length of the eye is about 15 to 17 mm and elongatesto approximately 23.6 to 24.0 mm in adults. Since most subjects in amostly agrarian society are emmetropic, a finely tuned feed-backmechanism which sensors the sharpness of the image of the retina,communicates with the effector part by handing over the information ofan axial length too short or too long, and governs the growth of theeye, in particular in its axial length, has to be present.

It is postulated that said finely tuned feed-back mechanism is locatedin the retina, since the retina is the only photosensitive part of theeye. Since the retinal photoreceptors as first element in the chain ofphotoreception and phototransduction are highly specialized (underexperimental conditions, a single photoreceptor can detect thephysically smallest unit of light energy, i.e., a photon), it isunlikely that the process of detecting haziness (defocus) of an image islocated primarily within the photoreceptors. The next following groupsof cells are the horizontal cells and amacrine cells in the innernuclear layer. They have been shown to be associated with contrastformation and enhancement of the information obtained by thephotoreceptors. The horizontal cells and amacrine cells form a densehorizontal network in the outer and inner plexiform layer of the retina.Since contrast is an essential part of detecting haziness of an image,it is likely that the horizontal/amacrine cells in association withretinal Muller cells, bipolar cells and other cells in the inner nuclearlayer of the retina form the primary sensory part of the feed-backmechanism controlling eye growth. An image is sharp if its borders aresharp, i.e., out of two neighboring photoreceptors, one is fullyactivated by the incoming light of the object's border, and the nextphotoreceptor is completely dark due to the sharp border of the image onthe retina. An image on the retina is blurred, if the decrease in theillumination of the photoreceptors at the border of the image decreasesin a slope-like fashion across several neighboring photoreceptors. Thesedifferences (slope-like configuration versus edge-like shape) can bedetected by the horizontal/amacrine cell system.

If the image of an object has been detected to be blurred (i.e., thelength of the optical axis of the eye is not in agreement with therefractive power of the optic system of the eye), the next step is todifferentiate whether the globe is too short (i.e., hyperopic) or toolong (i.e., myopic). This can be achieved by the chromatic aberration oflight within the eye: Short-wave length (blue) rays as compared tolong-wavelength (red) rays are physiologically diffracted to a largerextent and thus have a shorter optical axis. If now a white (i.e.,mixture of all spectral colors) image is fixed, its image will beprojected onto the retina. If the blue-sensitive photoreceptors convey asharper image than the red-sensitive photoreceptors do, the axial length(optical axis) is too short; if the red-sensitive photoreceptors conveya sharper image than the blue-sensitive photoreceptors do, the axiallength (optical axis) is too long. The system can thus differentiatewhether the axial growth of the eyes has to be increased or decreased.

The effector of the process of emmetropization is postulated to be theretinal pigment epithelium (RPE) with its underlying Bruch's membrane(BM). This is in contrast to common belief that the sclera as the outerthick coat of the eye is the determinant of the axial elongation of theglobe. In the course of a myopic axial elongation, the posterior scleramarkedly thins from about 1 mm in emmetropic eyes to 0.1 mm in highlymyopic eyes. In view of this marked thinning of the posterior sclera, ithas commonly been accepted that the sclera is the primary effector ofthe excessive axial elongation in high myopia and may thus beresponsible for the normal axial growth of the emmetropic eye. Thisassumption, however, is unlikely due to the following anatomical facts.In this context, FIGS. 1 and 2 provide an overview over the gross andmicroscopical anatomy of the eye.

If it were the sclera which primarily extends and makes the eye longer,the distance between the sclera and the inner layers, in particularBruch's membrane (BM), should enlarge. The space between the sclera andBM is filled-up with the choroid. With increasing axial myopia, thechoroid however gets markedly thinner (from about 250 pm to less than 30pm) and the distance between BM and the sclera profoundly decreases.This may suggest that in high axial myopia the inner parts of the eyeare not pulled outward (by an extending and elongating sclera), but arepushed outward by an internal process. Further, the sclera is verybradytrophic and contains only few nerves and blood vessels. It appearsunlikely that a finely tuned mechanism such as the ocularemmetropization in which the axial length has to be controlled with anaccuracy of less than 0.1 mm depends on such a little organizedstructure as the sclera. Also, the incoming light beams have to befocused onto the retinal photoreceptor outer segments very close to, orin, the RPE lying on the inner surface of BM. The sclera however, isseparated from the RPE layer by the spongy choroid the thickness ofwhich varies in dependence of factors such as daytime and presumablybody position and cerebrospinal fluid pressure. This makes itfurthermore unlikely that the sclera is the primary effector in theprocess of emmetropization.

In view of these findings, it is more likely that the RPE and BM formthe effector part of the process of emmetropization. BM consists of thebasal membrane of the RPE on its inner side, the basal membrane of thechoriocapillaris on its outer side, and two collagenous layers separatedfrom each other by an elastic layer in its center. Together with theRPE, BM forms the border between the choroid and retina, with theleaking choriocapillaris and the increased oncotic pressure on itschoroidal side and with the retina-blood barrier and mostly edema freeretinal tissue on its inner side. This watershed function of the BM isof high clinical importance since any defect in the BM-RPE complex leadsto a leakage of fluid into the retinal tissue and secondary disruptionof retinal function. BM is separated from the sclera by the looselyarranged spongy choroid and shows a physiologic opening as the innerpart of the optic nerve head. Besides its barrier function, BM hasbiomechanical properties since it is a continuous inner shell whichstretches from the ciliary body and indirectly from the scleral spur asits anterior anchoring to the peripapillary ring as its posteriorfixation. The biomechanical properties of BM have not been explored yet.In a similar manner, the active changes occurring in BM during thegrowth of the eye have not been examined yet. Since BM is the basalmembrane of the RPE, the RPE continuously produces BM material as is thecase for any basal membrane. If the sclera cannot be the effector partof the emmetropization process, the only structure remaining to fulfillthe function is the RPE/BM complex. The choroid itself is too spongy tobe able to exert any pressure, and in a similar manner the retina is toospongy and too highly specialized for photoreception, phototransduction,image processing, and eventually transport of the information to thevisual cerebral centers.

Thus, facts pointing to the RPE/BM complex as the primary effector forthe process of emmetropization (and myopization) are: BM is not thinnerin highly myopic eyes as compared to emmetropic eyes, the sclera howevergets markedly thinned in highly myopic eyes. Further, in eyes withsecondary high myopia due to congenital glaucoma, the myopic scleralthinning occurs also in the anterior segment of the eye in contrast toprimary high myopia in which only the posterior sclera thins.Furthermore, in eyes with secondary high myopia due to congenitalglaucoma, BM appears to be thinner than in eyes with primary myopia andthinner than in emmetropic eyes, suggesting that in secondary highmyopia the increased elevated intraocular pressure (due to congenitalglaucoma) within the first two years of life (during which thesclera—according to the existing model of thinking—or BM and thesclera—according to the mechanism described above—is passively stillexpandable) leads to a passive elongation of the ocular coats. Moreover,beyond an age of about 2 to 3 years, the scleral volume no longerincreases and is then independent of the axial length. In contrast,scleral thickness, the more the closer to the posterior pole of the eye,decreases with longer axial length. This suggests that axial elongationbeyond the physiological growth of the eye occurs by a rearrangement ofthe sclera and not by active scleral growth and increase in scleralvolume. Finally, in a similar manner, beyond an age of about 2 to 3years, the choroidal volume does not increase and is then independent ofaxial length. In contrast, choroidal thickness, the more the closer tothe posterior pole of the eye, decreases with longer axial length. Thissuggests that axial elongation beyond the physiological growth of theeye occurs by a rearrangement of the choroid and not by active choroidalgrowth and increase in choroidal volume.

The RPE governs the axial elongation by the formation of new basalmembrane material leading to the elongation and growth of BM. If, aspointed out above, the horizontal cells/amacrine cells in associationwith retinal Muller cells, bipolar cells and other cells in the innernuclear layer of the retina are the sensory part of the process ofemmetropization and myopization, and if the RPE/BM complex is theeffector part, the question is how both parts communicate with eachother. Since the transferal of such information by nerves is highlyunlikely in the retina (nerves have not been found yet, short distancebetween the sensory part (horizontal cells/amacrine cells) and theeffector part (RPE), potential continuous flow of fluid from theintraretinal space through the RPE/BM complex into the choroid), thesignal transduction might take place by substances such as cytokines,chemokines, growth factors or similar proteins.

In view of the above, the technical problem underlying the presentinvention is to identify mediators of signal transduction betweenhorizontal cells/amacrine cells and the RPE/BM complex, and, based onsuch findings, to provide agents that can be used in the prophylactic ortherapeutic treatment of myopia and hyperopia.

The solution to the above technical problem is achieved by theembodiments characterized in the claims.

In particular, in a first aspect, the present invention relates to anagent that is capable of decreasing epidermal growth factor receptor(EGFR) signaling and/or signaling of another receptor susceptible foramphiregulin in a subject in a direct or indirect manner, for use in theprophylactic or therapeutic treatment of myopia in said subject.

Preferably, said agent is selected from the group consisting of:

-   -   (a) an isolated antibody, antibody fragment or antibody mimetic        directed against native amphiregulin in said subject;    -   (b) an isolated antibody, antibody fragment or antibody mimetic        directed against a native amphiregulin precursor in said        subject;    -   (c) an isolated antibody, antibody fragment or antibody mimetic        directed against EGFR in a blocking manner in said subject;    -   (d) an isolated antibody, antibody fragment or antibody mimetic        directed against another receptor susceptible for amphiregulin        in a blocking manner in a subject;    -   (e) an EGFR inhibiting agent; and    -   (f) a small interfering RNA (siRNA) agent capable of reducing        the expression of amphiregulin in a subject.

Amphiregulin, also known as AREG, is a protein that in humans is encodedby the AREG gene. It is a member of the epidermal growth factor family,and is an autocrine growth factor as well as a mitogen for astrocytes,Schwann cells and fibroblasts. It is related to epidermal growth factor(EGF) and transforming growth factor alpha (TGF-alpha). Further, itusually interacts with the EGF receptor (EGFR) to promote the growth ofnormal epithelial cells, as well as with other receptors susceptible foramphiregulin. In the genome-wide joint meta-analysis underlying thepresent invention (cf. the data provided in the Examples of the presentapplication), the AREG gene displayed the strongest correlation withrefractive error. Further, amphiregulin is not a neurotransmitter (incontrast to e.g. gamma amino butyric acid), so that the secretion ofamphiregulin does not interfere with the normal signal transduction inthe visual afference. Furthermore, as a member of the family ofepidermal growth factors, amphiregulin has an effect on epithelial cellssuch as the RPE. Based on these findings, amphiregulin has beenidentified in the present invention as a key mediator of axialelongation of the eye, due to its function as a signaling moleculebetween the sensory network in the middle retinal layers and the RPE/BM.

The term “decreasing epidermal growth factor receptor (EGFR) signalingand/or signaling of another receptor susceptible for amphiregulin in asubject in a direct or indirect manner” indicates the capability of theagents of the present invention to reduce the activity of the downstreamsignaling pathways of the respective receptors, either by a directinteraction with the receptors, as is e.g. the case for an antibody,antibody fragment or antibody mimetic directed against EGFR in ablocking manner, or by an indirect mechanisms, e.g. the inactivation ofamphiregulin by a suitable antibody, antibody fragment or antibodymimetic directed against native amphiregulin, or the reduction ofamphiregulin expression by a suitable siRNA.

In preferred embodiments, signaling is completely blocked, or is reducedby at least 10%, at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 92.5%, at least 95%, at least 97%, at least 98%, atleast 98,5%, at least 99%, at least 99.25%, at least 99.5%, or at least99.75% as compared to normal physiologic levels.

The term “isolated antibody or antibody fragment” as used herein relatesto isolated, i.e., purified, native poly- or monoclonal antibodies orrecombinantly produced poly- or monoclonal antibodies and fragmentsthereof. Antibody fragments are preferably selected from the groupconsisting of Fab fragments, F(ab′)₂ fragments and Fab′ fragments.Methods for the isolation and/or purification of native antibodies arenot particularly limited and are known in the art. Further, methods forthe generation and expression of recombinant antibodies or antibodyfragments are not particularly limited and are known in the art.Furthermore, methods for the generation of antibody fragments are notparticularly limited and are known in the art.

The term “antibody mimetic” as used herein relates to proteinaceouscompounds that, like antibodies, are capable of binding to a giventarget structure in a specific manner. Preferably, antibody mimetics areselected from the group consisting of single-chain variable fragments(scFv), single-domain antibodies, affibodies, affilins, affimers,affitins, anticalins, DARPins, monobodies, and peptide aptamers.

Respective antibody mimetics and methods for producing the same are notparticularly limited and are known in the art.

The term “antibody, antibody fragment or antibody mimetic directedagainst native amphiregulin/a native amphiregulin precursor/EGFR/anotherreceptor susceptible for amphiregulin in a subject” as used hereinindicates the ability of said antibodies, antibody fragments or antibodymimetics to bind to native amphiregulin, to a native amphiregulinprecursor, to the EGFR, or to another receptor susceptible foramphiregulin in a subject in a specific manner, i.e., to recognize andbind a specific epitope of said amphiregulin, amphiregulin precursor,EGFR, or other receptor susceptible for amphiregulin. In this context,the term “antibody directed against amphiregulin/an amphiregulinprecursor/EGFR/another receptor susceptible for amphiregulin” as usedherein is intended to be equivalent to the term“anti-amphiregulin/anti-amphiregulin precursor/anti-EGFR/anti-receptorantibody”.

Amphiregulin precursors are known in the art and includepro-amphiregulin, wherein the latter is a preferred amphiregulinprecursor. Further, receptors susceptible for amphiregulin besides EGFRare known in the art.

The term “antibody, antibody fragment or antibody mimetic directedagainst EGFR/another receptor susceptible for amphiregulin in a blockingmanner in a subject” as used herein indicates the ability of saidantibodies, antibody fragments or antibody mimetics to inhibit theactivation of EGFR or of another receptor susceptible for amphiregulinby amphiregulin in a direct or indirect manner, e.g. by stericallyprohibiting the binding of amphiregulin to the EGFR or said otherreceptor or by inhibiting downstream effector functions of EGFR or saidother receptor.

Antibodies, antibody fragments or antibody mimetics that are directedagainst native amphiregulin, e.g. native human amphiregulin, are notparticularly limited and are known in the art. Further, antibodies thatare directed against EGFR, e.g. human EGFR, in a blocking manner are notparticularly limited and are known in the art. The same applies toantibodies, antibody fragments or antibody mimetics that are directedagainst amphiregulin precursors or against other receptors susceptiblefor amphiregulin in a blocking manner.

EGFR inhibiting agents for use in the present invention are notparticularly limited and are known in the art. Respective agents arepreferably selected from the group consisting of monoclonal antibodyinhibitors of EGFR and small molecules to inhibit the EGFR tyrosinekinase.

Small interfering RNA (siRNA) agents and methods for producing and usingthe same are not particularly limited and are known in the art.According to the present invention, siRNA agents are capable of reducingthe expression of amphiregulin in a subject. In preferred embodiments,the antisense strand of such siRNA agents, i.e., the strand targetingamphiregulin mRNA, consists of one of the sequences shown in SEQ ID NO:2 and 3.

For use in the prophylactic or therapeutic treatment of myopia accordingto the present invention, the agent is preferably administeredintravitreally, epicorneally, transcorneally, transsclerally,transconjunctivally, subconjunctivally, intraocularly, or systemically(e.g. orally, rectally, or intravenously). Respective methods foradministration, as well as doses and dosage regimens are notparticularly limited and are known in the art. Typical doses for anindividual administration of e.g. antibodies, antibody fragments orantibody mimetics are in the range of 1 to 2 mg. As for e.g.intravitreal administration, the injected volume is typically in therange of 50 to 200 μl.

In a preferred embodiment, the subject to be treated is a human subject.

In particularly preferred embodiments according to the first aspect ofthe present invention, the agent is an antibody, antibody fragment orantibody mimetic directed against (i) native amphiregulin in a subject,or (ii) the epidermal growth factor receptor (EGFR) in a blocking mannerin a subject.

In a second aspect, the present invention relates to an agent that iscapable of increasing epidermal growth factor receptor (EGFR) signalingand/or signaling of another receptor susceptible for amphiregulin in asubject, for use in the prophylactic or therapeutic treatment ofhyperopia in said subject.

Preferably, said agent is selected from the group consisting of:

-   -   (a) isolated amphiregulin or a biologically active fragment or        derivative thereof;    -   (b) an isolated amphiregulin precursor or a biologically active        fragment or derivative thereof;    -   (c) an isolated antibody, antibody fragment or antibody mimetic        directed against the epidermal growth factor receptor (EGFR) in        an activating manner in said subject;    -   (d) an isolated antibody, antibody fragment or antibody mimetic        directed against another receptor susceptible for amphiregulin        in an activating manner in a subject; and    -   (e) an EGFR activating agent.

The term “increasing epidermal growth factor receptor (EGFR) signalingand/or signaling of another receptor susceptible for amphiregulin in asubject” indicates the capability of the agents of the present inventionto increase the activity of the downstream signaling pathways of therespective receptors.

In preferred embodiments, signaling is increased by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, or more.

The term “isolated amphiregulin/amphiregulin precursor” as used hereinrelates to isolated, i.e., purified amphiregulin/amphiregulin precursoror recombinantly produced amphiregulin/amphiregulin precursor. Methodsfor the isolation and/or purification of nativeamphiregulin/amphiregulin precursor are not particularly limited and areknown in the art. Further, methods for the generation and expression ofrecombinant amphiregulin/amphiregulin precursor are not particularlylimited and are known in the art.

Amphiregulin precursors are known in the art and includepro-amphiregulin, wherein the latter is a preferred amphiregulinprecursor. Further, receptors susceptible for amphiregulin besides EGFRare known in the art.

In preferred embodiments, said amphiregulin comprises or preferablyconsists of the amino acid sequence shown in SEQ ID NO: 1.

In this context, the term “biologically active fragment ofamphiregulin/amphiregulin precursor” as used herein relates to fragmentsof amphiregulin/amphiregulin precursor that have one or more amino aciddeletions with respect to native amphiregulin/amphiregulin precursor,while at the same time retaining the biological activity of binding tothe EGF receptor or another receptor susceptible for amphiregulin andactivating the same. Further, the term “derivative ofamphiregulin/amphiregulin precursor” as used herein relates topolypeptides that are derived from native amphiregulin/amphiregulinprecursor but have one or more amino acid substitutions, additions, ordeletions with respect to native amphiregulin while at the same timeretaining the biological activity of binding to the EGF receptor oranother receptor susceptible for amphiregulin and activating the same.In this context, methods for determining the biological activity ofamphiregulin or amphiregulin precursors are not particularly limited andare known in the art.

While the number of amino acid substitutions and/or additions and/ordeletions is generally only limited by the above proviso concerning thebiological activity of the resulting amphiregulin fragment oramphiregulin derivative polypeptide, it is preferable that the resultingfragment or polypeptide has at least 50%, at least 52.5%, at least 55%,at least 57.5%, at least 60%, at least 62.5%, at least 65%, at least67.5%, at least 70%, at least 72.5%, at least 75%, at least 76.25%, atleast 77.5%, at least 78.75%, at least 80%, at least 81.25%, at least83.75%, at least 85%, at least 86.25%, at least 87.5%, at least 88%, atleast 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%,at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, atleast 95.25%, at least 95.5%, at least 95.75%, at least 96%, at least96.25%, at least 96.5%, at least 96.75%, at least 97%, at least 97.25%,at least 97.5%, at least 97.75%, at least 98%, at least 98.25%, at least98.5%, at least 98.75%, at least 99%, at least 99.25%, or at least99.5%, identity to native amphiregulin, e.g. native human amphiregulinaccording to SEQ ID NO: 1.

The terms “isolated antibody or antibody fragment” and “antibodymimetic” as used for this second aspect of the present invention are asdefined above for the first aspect of the present invention. The sameapplies for the term “antibody, antibody fragment or antibody mimeticdirected against EGFR/other receptor susceptible for amphiregulin”.

The term “antibody, antibody fragment or antibody mimetic directedagainst EGFR/other receptor susceptible for amphiregulin in anactivating manner in a subject” as used herein indicates the ability ofsaid antibodies, antibody fragments or antibody mimetics to activateEGFR or another receptor susceptible for amphiregulin independently ofamphiregulin, i.e., to trigger downstream effector functions of EGFR orsaid other receptor susceptible for amphiregulin.

Antibodies, antibody fragments or antibody mimetics that are directedagainst EGFR, e.g. human EGFR, in an activating manner are notparticularly limited and are known in the art. The same applies toantibodies, antibody fragments or antibody mimetics that are directedagainst other receptors susceptible for amphiregulin in an activatingmanner.

EGFR activating agents for use in the present invention are notparticularly limited and are known in the art. Respective agents arepreferably selected from the group consisting of members of theepidermal growth factor (EGF) family (including transforming growthfactor-α (TGF-α), amphiregulin (AR), epiregulin (EPR), epigen,betacellulin (BTC), neuregulin-1 (NRG1), neuregulin-2 (NRG2)neuregulin-3 (NRG3), and neuregulin-4 (NRG4)).

For use in the prophylactic or therapeutic treatment of hyperopiaaccording to the present invention, the agent is preferably administeredintravitreally, epicorneally, transcorneally, transsclerally,transconjunctivally, subconjunctivally, intraocularly, or systemically(e.g. orally, rectally, or intravenously). Respective methods foradministration, as well as doses and dosage regimens are notparticularly limited and are known in the art. Typical doses for anindividual administration of e.g. antibodies, antibody fragments orantibody mimetics, or of amphiregulin are in the range of 1 to 2 mg. Asfor e.g. intravitreal administration, the injected volume is typicallyin the range of 50 to 200 μl.

In a preferred embodiment, the subject to be treated is a human subject.

In particularly preferred embodiments according to the second aspect ofthe present invention, the agent is isolated amphiregulin or abiologically active fragment or derivative thereof, or an isolatedantibody, antibody fragment or antibody mimetic directed against theepidermal growth factor receptor (EGFR) in an activating manner in asubject.

In a further aspect, the present invention relates to a method for theprophylactic or therapeutic treatment of myopia in a subject, comprisingthe step of administering a therapeutically effective amount of an agentas defined for the first aspect of the present invention to saidsubject. In yet a further aspect, the present invention relates to amethod for the prophylactic or therapeutic treatment of hyperopia in asubject, comprising the step of administering a therapeuticallyeffective amount of an agent as defined for the second aspect of thepresent invention to said subject.

In both of these aspects, the (i) agents, (ii) antibody, antibodyfragment or antibody mimetic directed against native amphiregulin, (iii)antibody, antibody fragment or antibody mimetic directed against anative amphiregulin precursor, (iv) antibody, antibody fragment orantibody mimetic directed against EGFR in a blocking manner, (v)antibody, antibody fragment or antibody mimetic directed against anotherreceptor susceptible to amphiregulin in a blocking manner, (vi)amphiregulin or a biologically active fragment or derivative thereof,(vii) amphiregulin precursor or a biologically active fragment orderivative thereof, (viii) antibody, antibody fragment or antibodymimetic directed against the epidermal growth factor receptor (EGFR) inan activating manner, (ix) antibody, antibody fragment or antibodymimetic directed against another receptor susceptible for amphiregulinin an activating manner, (x) EGFR inhibiting agent, (xi) EGFR activatingagent, (xii) siRNA agent, (xiii) administration, and (xiv) subject areas defined for the first two aspects of the present invention. Further,respective therapeutically effective amounts are known in the art.

In a further aspect, the present invention relates to a method for thediagnosis of myopia or hyperopia, or of a predisposition for thedevelopment of myopia or hyperopia, in a subject, comprising the stepsof:

-   -   (a) providing a biological sample from the subject;    -   (b) determining the amphiregulin level in said sample;    -   (c) comparing the level determined in step (b) to the        amphiregulin levels found in emmetropic subjects or subjects        going to be emmetropic; and    -   (d) determining that the subject has myopia or is predisposed        for the development of myopia in case the level determined in        step (b) is higher than the amphiregulin levels found in        emmetropic subjects or subjects going to be emmetropic; and        determining that the subject has hyperopia or is predisposed for        the development of hyperopia in case the level determined in        step (b) is lower than the amphiregulin levels found in        emmetropic subjects or subjects going to be emmetropic.

Types of biological samples for practicing this method are known to aperson skilled in the art and include blood, blood serum, blood plasma,and cerebrospinal fluid. Further, methods for determining theamphiregulin level in a biological sample are not particularly limitedand are known in the art.

In a final aspect, the present invention relates to a method foridentifying agents which associate with amphiregulin or fragments orvariants thereof, comprising the steps of:

-   -   (a) contacting said amphiregulin or fragments or variants        thereof, or a recombinant host cell expressing amphiregulin or        fragments or variants thereof, with a test compound and    -   (b) analyzing the ability of said test compound to bind to        amphiregulin or fragments or variants thereof.

In preferred embodiments of this method, the ability of said testcompound to bind to amphiregulin or a fragment or variant thereof or theability of said test compound to inhibit binding of said amphiregulin ora fragment or variant thereof to EGFR or any other receptor susceptiblefor amphiregulin or a variant thereof is analyzed.

In further preferred embodiments, said method further comprises the stepof formulating said compound or a pharmaceutically acceptable saltthereof with one or more pharmaceutically acceptable excipient(s) and/orcarrier(s).

In this context, methods for generating recombinant host cell expressingamphiregulin or fragments or variants thereof, respective amphiregulinfragments or amphiregulin variants, methods for analyzing the ability ofsaid test compound to bind to amphiregulin or fragments or variantsthereof, and methods for analyzing the ability of said test compound toinhibit binding of said amphiregulin or a fragment or variant thereof toEGFR or any other receptor susceptible for amphiregulin or a variantthereof, are not particularly limited and are known in the art.

In the genome-wide joint meta-analysis underlying the present invention(cf. the data provided in the Examples of the present application),further genes and genetic markers that correlated with refractive errorin addition to amphiregulin have been identified. These are (i) FAM150B(family with sequence similarity 150 member 2) which interacts withleukocyte tyrosine kinase (LTK), (ii) LINC00340, (iii) FBN1 (fibrillin1), (iv) MAP2K1 (mitogen-activated protein kinase 1) which interactswith muscarinic receptors, (v) ARID2 (AT-rich interactive domain 2)which is a subunit of the chromatin remodeling complex PBAF, (vi)SLC14A2 (solute carrier family 14 member 2) which is an ureatransporter, (vii) GABRR1 which is a receptor for gamma-aminobutyricacid (GABA), and (viii) PDE10A which is an enzyme that can hydrolyzecAMP/cGMP. These can be used in the present invention in an analogousmanner to amphiregulin as described herein.

The present invention is based on the finding of a mechanism in thedevelopment of myopia as state-out-of-control of the physiologicalprocess of emmetropization of the eye. Emmetropization of the eye refersto the process of controlled increase in the ocular dimensions (axiallength, horizontal and vertical globe diameters, curvature of theanterior and posterior lens surface, position of the lens, curvature ofthe anterior and posterior surface of the cornea, refractive indices inthe various optic media, distances between the various elements of theoptic media of the eye). The mechanism includes a sensory part whichdetects that the image of objects on the retina is not sharply focusedand blurry. The detection of a defocused image will be in the layer ofhorizontal cells and amacrine cells in the middle layers of the retina.The network provided by the intertwining of the photoreceptor axons, theaxons and dendrites of the horizontal and amacrine cells and bipolarcells, in close association with the retinal Muller cells, will detectwhether the edges of the images are sharp or blurry. This process maypartially be associated with contrast sensitivity and contrastenhancement or contrast suppression physiologically taking place inthese retinal layers.

If the sharpness or haziness of the image edges has been detected, thecells in the intertwining network will also sensor whether the edges forblue images or for red images are sharper, since light of blue color,green color and red color is sensed by three different types of conephotoreceptors. Using the physical principle of chromatic aberration, asharp image in the blue range instead of the red range indicates thatthe axial length of the eye is too short, whereas a sharp image in thered range instead of the blue range indicates that the axial length ofthe eye is too long. Correspondingly, in the case of a sharp blue imageand defocused red image, the network of cells will give a signal to theeffector element in the process emmetropization of the eye to increasethe ocular axial length. In the case of a sharp red image and defocusedblue image, the network of cells will give a signal to the effectorelement in the process emmetropization of the eye to stop the increasein ocular length or to decrease the speed in increasing the axial lengthduring the physiological growing of the eye.

Structures in the growing eye, which are involved in governing axialelongation during the physiological growth of the eye are the retinalpigment epithelium cells (RPE), which are close enough to the retinalnetwork in the middle retinal layers to receive signals, are pluripotentdue to their relatively low degree of specialization duringembryogenesis, and above all form the inner part of Bruch's membrane(BM) as their basal membrane. BM can be regarded as a biomechanicallyimportant inner shell in the eye, connected to the pars plicata of theciliary body with the ciliary muscle and indirectly to the scleral spurin the anterior segment, and to the peripapillary ring of the opticnerve head in the posterior segment. If the network in the middleretinal layers gives a signal to the RPE cells to produce more basalmembrane, this results in the deposition of more basal membrane materialin the inner layer of BM which subsequently will elongate. Elongation ofBM means that BM pushes the sclera more outward by transmitting thegrowth-related pressure through the spongy choroid onto the sclera.Further, it secondarily leads to a thinning of the choroid.Correspondingly, axial elongation after an age of 2 to 3 years (i.e.,after the end of the physiological growth of the globe) is associatedwith a thinning of the sclera and choroid, however it is not associatedwith an increase in the volume of sclera and choroid, contradicting anactive role of both tissues in the process of emmetropization. As acorollary, BM, in contrast to the choroid and sclera, does not decreasein thickness in axial elongation pointing to the active role of BM inshaping and fine-tuning the final shape and length of the eye.

The signaling pathway between the sensory network in the middle retinallayers and the RPE/BM as the effector part uses molecules such ascytokines, chemokines, growth factors or similar proteins, which eithergive an impulse to the RPE to lay down more basal membrane material orto decrease the production of this material. In this context,amphiregulin has been identified in the present invention as one ofthese molecules and as a key mediator of axial elongation of the eye.

The figures show:

FIG. 1:

Gross Anatomy of the Eye

Vertical sagittal section of the adult human eye.

FIG. 2:

Microscopical Anatomy of the Eye

Organization of the retina in a schematic vertical section. Interneuronsare designates as (B) bipolar cells, (A) amacrine cells, (H) horizontalcells, and (IP) interplexiform cells. In reality, the retina is packedwith cells, and there is hardly any extracellular space. The ten layersof the retina are listed on the right-hand side of the figure: (1)Pigment epithelium of retina, (2) layer of rods and cones (outersegments of photoreceptors), (3) outer limiting membrane (end-feet ofMuller cells), (4) layer of nuclei of photoreceptors, (5) outerplexiform layer (synapses), (6) inner nuclear layer (somata ofinterneurons and nuclei of Muller cells), (7) inner plexiform layer(synapses), (8) ganglion cell layer, (9) nerve fiber layer, (10) innerlimiting membrane (end-feet of Muller cells). The potential spacebetween the pigment epithelium and the layer of rods and cones is the“optic ventricle”, the remnant of the cavity of the optic vesicle.

FIG. 3:

Manhattan Plots of −log₁₀(P) for the Joint Meta-Analysis on SNP andSNP×Education Effects on Spherical Equivalent in A) European AncestryPopulations and B) Asian Population

The upper horizontal dashed line indicates the genome-wide significancelevel of p<5×10⁻⁸. The lower horizontal dashed line indicates thesuggestive significance level of p<1×10⁻⁵. Only novel loci reachinggenome-wide significance are labeled.

The present invention discloses the following sequences:

Human amphiregulin amino acid sequence SEQ ID NO: 1MRAPLLPPAP VVLSLLILGS GHYAAGLDLN DTYSGKREPF SGDHSADGFE VTSRSEMSSG  60SEISPVSEMP SSSEPSSGAD YDYSEEYDNE PQIPGYIVDD SVRVEQVVKP PQNKTESENT 120SDKPKRKKKG GKNGKNRRNR KKKNPCNAEF QNFCIHGECK YIEHLEAVTC KCQQEYFGER 180CGEKSMKTHS MIDSSLSKIA LAAIAAFMSA VILTAVAVIT VQLRRQYVRK YEGEAEERKK 240LRQENGNVHA IA 252siRNA antisense strand targeting human amphiregulin mRNA SEQ ID NO: 2CGAAC CACAA AUACC UGGCT TsiRNA antisense strand targeting human amphiregulin mRNA SEQ ID NO: 3CCUGG AAGCA GUAAC AUGCT T

The present invention will be further illustrated by the followingexamples without being limited thereto.

EXAMPLES

Experimental Procedures

Study Populations

From the Consortium of Refractive Error and Myopia (CREAM), a total of34 studies comprising 40,036 individuals of European ancestry from 25studies and 10,315 individuals of Asian ancestry from 9 studies wererecruited. Individuals aged less than 20 years old were excluded, aswell as those who had undergone cataract surgery, laser or otherintra-ocular procedures that could alter refraction. Many of thesestudies were also included in the previous CREAM genome-wide associationstudy (GWAS) on spherical equivalent. All studies adhered to the tenetsof the Declaration of Helsinki and were approved by their local researchethics committees. All participants provided a written, informed consentbefore the start of the study.

Phenotyping and Education Levels

Participants in the included studies underwent complete ophthalmologicalexamination. Non-dilated refraction was measured by auto-refractionand/or subjective refraction. Spherical equivalent was calculated as thesphere power plus half of the cylinder power for each eye. The meanspherical equivalent of the right and left eyes was used as aquantitative outcome. When data from only one eye was available, thespherical equivalent of that eye was used. For education, subjectsreported the highest level of education achieved, or the years ofschooling through a self-reported questionnaire or in an interview.

Education for all participants was dichotomized into a higher educationgroup consisting of those who had completed at least higher secondaryeducation, polytechnic, or ≥12 years spent in formal education, and alower education group including individuals who had only completed lowersecondary education or less, or with <12 years of formal education. Infour cohorts of relatively young European participants (total samplesize of 2,349), almost all had completed 12 or more years of schooling.It was thus chosen to categorize individuals with tertiary or universityeducation as the higher educaiton group. Sensitivity analysis excludingthese four cohorts did not appreciably change meta-analysis results.

Genotyping and Imputation

Each study applied stringent quality control filters for GWAS. Ingeneral, individuals reflecting duplicates, low call rate (<95%), gendermismatch, or population outliers we excluded. SNPs were excluded incases of low genotyping call rate (>5% missingness), monomorphic SNPs,with MAF <1%, or in Hardy-Weinberg disequilibrium (p-value <10⁻⁶). Afterquality control (QC) filtering, the array genotypes of each study wereimputed using the 1000 Genomes Project data as reference panels (build37, phase 1 release, March 2012) with the software Minimac or IMPUTE.SNPs which passed imputation quality thresholds (MACH: r²>0.5 or IMPUTEinfo score >0.5) and with minor allele frequency≥5% were carried forwardfor the meta-analysis.

Statistical Models

For each study, a linear regression model at each genotyped or imputedSNP was constructed, with the mean spherical equivalent as the outcome.An additive genetic model was assumed where the number of risk allelesis an ordinal variable (0, 1 and 2) for directly genotyped SNPs, or acontinuous variable of allele dosage probability ranging from 0 to 2 forimputed SNPs. The primary analytic model included SNP, education, aSNP×education interaction term, as well as age and sex as covariates.Additional adjustments for the top principal components of genomicmarker variations were performed in individual studies when applicable(i.e., when there was evidence of population stratification).

The following additive genetic model was used to test for a joint effectof SNP (β_(SNP)) and SNP×education interaction (β_(SNP×education)) onmean spherical equivalent:

Y=β₀+β_(SNP)×SNP+β_(education)×Education+β_(SNP×education)×SNP×Education+β_(c)×Cov+ε  (Model1)

where Y is the mean spherical equivalent, education is a dichotomousvariable (0=lower education group and 1=higher education group); Cov isa set of covariates such as age, sex and first top five principalcomponents when applicable. For family-based studies, the kinship matrixwas estimated empirically from the SNP data and included as a randomeffect in the generalized mixed model. To test an effect ofSNP×education interaction, β_(SNP×education) was assessed from Model 1.

The linear regression analyses in each study were conducted withQuickest (http://toby.freeshell.org/software/quicktest.shtml) orProbABEL (http://www.genabel.org/packages/ProbABEL) for the unrelatedsamples, and MixABEL (http://www.genabel.org/packages/MixABEL) forfamily-based data. The command ‘robust’ was used in the above softwareto calculate the robust (‘sandwich’, Huber-White) standard errors ofβ_(SNP) and β_(SNP×education), and error covariance of βs, to correctthe potential inflation of false positive rate for the interactionp-value.

In addition, each study also tested the main effect of education onspherical equivalent by adjusting for age and gender using the linearregression model:

Y=β₀+β_(education)×education+β_(c)×Cov+ε  (Model 2)

where the definition of the variables is the same as in Model 1.

GWAS Meta-Analyses

The joint meta-analysis (JMA) approach was adopted to simultaneouslytest both main SNP effects and SNP×education interactions for sphericalequivalent with a fixed-effect model, using SNP and SNP×educationregression coefficients and a betas' covariance matrix from each study.A Wald statistic, following a chi-square distribution with two degreesof freedom, was used to test the joint significance of the SNP andSNP×education regression coefficients. The JMA was performed with METAL(http://www.sph.umich.edu/csg/abecasis/metal/), using a script patch. ACochran's Q test was used to assess heterogeneity of the betacoefficients across studies for the SNP and interaction effects. To testfor interaction between the SNP and education, a secondary meta-analysisof the SNP×education interaction effects for spherical equivalent (onedegree of freedom) with a fixed-effects model using inverse-varianceweighting in METAL was conducted; this is a traditional meta-analysis toinvestigate SNP×education interactions per se. Effects and standarderror of the SNP (β_(SNP)) on spherical equivalent in the lowereducation group and higher education (β_(SNP)+β_(SNP×education)) werederived from the JMA output.

A meta-regression was performed to explore sources of heterogeneity inthe meta-analysis for three loci showing G×E interactions (R package‘metafor’; http://www.rproject.org/). Meta-regression included thefollowing study-specific variables as covariates: study sample size,proportion of individuals in the higher education group, averagespherical equivalent, education main effects, ethnicity, study design,study year, and average age.

The study-specific genomic control inflation factors λ_(gc) for thejoint test for SNP and interaction term ranged from 1.009 to 1.125 withan average of 1.019, calculated by the ratio of the observed medianchi-square divided by the expected median of the 2df chi-squaredistribution (1.382). Genomic control (GC) correction was applied tochi-square statistics in each individual study. For three studies ofsmall sample sizes (N<500) and λ_(gc) greater than 1, SNPs showingsignificant joint P value <1×10⁻⁵ were further excluded prior tostarting the meta-analysis, but neither the main effects nor theinteraction effects supporting such an association. Quantile-quantile(QQ) plots of the p-values showed only modest inflation of the teststatistics in the JMA (Europeans: λ_(meta)=1.081; Asians:λ_(meta)=1.053; Combined: λ_(meta)=1.092, similar to previousgenome-wide JMA studies with comparable sample sizes. A small number ofmarkers in the meta-analysis with P_(HET)<0.0001 was excluded. Theλ_(gc) for the SNP×education interaction term in the individual studiesranged from 1.01 to 1.08, indicating little evidence of test statisticinflation for each study.

Annotation of Genetic Variants

The coordinates and variant identifiers are reported on the NCBI B37(hg19) genome build, and annotated using UCSC Genome Browser. Variantswithin each of the linkage disequilibrium (LD) blocks (r²>0.8) inEuropean and Asian populations of the 1000 Genomes Project (100 Kbflanking the top SNP; hg19) were identified to apply functionalannotations with experimental evidence of transcription regulation usingHaploReg (http://www.broadinstitute.org/mammals/haploreg/haploreg.php)and Encyclopedia of DNA Elements (ENCODE) data.

Gene Expression in Human Tissues GWAS Mets-Analyses and SNP FunctionAnnotation

To assess gene expression in human tissues, the Ocular Tissue Database(https://genome.uiowa.edu/otdb) and the EyeSAGE database were examined.The estimated gene and exome level abundances are available online.Normalization of gene expression used the Probe Logarithmic IntensityError (PLIER) method with GC-background correction.

Example 1 Education and its Main Effects on Spherical Equivalent

Baseline characteristics of 50,351 participants from 34 studies in ourmeta-analysis show that a total of 40,036 of subjects were of Europeanancestry and 10,315 were of Asian ancestry; the age of the participantsranged from 20 to 99 years. Among Europeans, the proportions ofparticipants who completed higher secondary education ranged from 16.0%to 94.4% with an average of 50.7%. In Asians, the proportions ofindividuals who completed higher secondary education ranged from 6.7% to75.9% with an average of 30.0%. Across all studies, individuals in thehigher education group had a spherical equivalent refractive error thatwas on average 0.59 diopters (D) more myopic, or less hyperopic,compared to those in the lower education group (β=−0.59; 95% Cl: −0.64,−0.55). High education level was associated with a two-fold more myopicspherical equivalent in individuals of Asian as compared to Europeanancestry (Asians: β=−1.09, 95% Cl: −1.20, −0.98; Europeans: β=−0.49, 95%Cl: −0.54, −0.44.

Example 2 Joint Meta-Analysis in Europeans

The genome-wide joint analysis for SNP main effect and SNP×educationinteraction by JMA in 40,036 European Ancestry individuals showedassociation with spherical equivalent at 9 previously implicated loci.Further, 4 previously unreported loci associated with sphericalequivalent were also identified achieving genome-wide significance (Pfor JMA <5.0×10⁻⁸; Table 1 and FIG. 3 A): FAM150B, LINC00340, FBN1, andDIS3L-MAP2K1. Two of them (FAM150B and DIS3L-MAP2K1) were replicated inAsians (P for JMA <0.05; refer to the following section). Littleevidence of heterogeneity in JMA was noted across studies (Q test:P_(het)≥0.086). The significant association for JMA at these loci inEuropeans was primarily due to SNP effects in both lower and highereducation strata (4.40×10⁻⁸≤P≤1.35×10⁻⁶, 7.61×10⁻¹¹≤P≤1.75×10⁻⁶,respectivelly). SNP×education interaction was not significant (P forinteraction≥0.208). The estimated effect sizes of SNP effects onspherical equivalent were highly similar across education strata.

TABLE 1 Six genetic loci associated with spherical equivalent from thejoint meta-analysis in the European populations and combined analysis.Europeans Asians All SNP (n = 40,306) (n = 10,315) (n = 50,351) (Chr:BP) Gene Allele FREQ Subgroup β P P_(het) β P P_(het) β P P_(het)rs60843830 FAM150B C/G 0.66/0.74 JMA 3.71 × 10⁻⁸ 0.086 0.0131 0.980 1.27× 10⁻⁹ 0.395 (2: 286756) Lower educaton −0.11 4.73 × 10⁻⁸ −0.09 0.010−0.10 1.65 × 10⁻⁹ Higher education −0.09 1.75 × 10⁻⁶ −0.06 0.509 −0.099.83 × 10⁻⁷ rs10946507 LINC00340 A/G 0.47/0.36 JMA 3.07 × 10⁻⁸ 0.2130.433 0.396 2.24 × 10⁻⁸ 0.249 (6: Lower educaton −0.08 7.08 × 10⁻⁷ −0.040.313 −0.08 6.13 × 10⁻⁷ 22100367) Higher education −0.09 1.19 × 10⁻⁸−0.08 0.450 −0.09 1.20 × 10⁻⁸ rs8023401 FBN1 G/A 0.83/0.85 JMA 1.66 ×10⁻⁹ 0.180 0.572 0.979 2.85 × 10⁻⁹ 0.495 (15: Lower educaton −0.15 4.40× 10⁻⁸ −0.06 0.304 −0.13 8.17 × 10⁻⁸ 48703823) Higher education −0.167.61 × 10⁻¹¹ −0.03 0.828 −0.14 2.02 × 10⁻⁹ rs16949788 DIS3L- T/C0.91/0.94 JMA 1.34 × 10⁻⁸ 0.721 0.0042 0.219 2.19 × 10⁻⁸ 0.245 (15:MAP2K1 Lower educaton −0.15 1.35 × 10⁻⁶ 0.21 0.103 −0.13 4.88 × 10⁻⁶66590037) Higher education −0.17 1.89 × 10⁻⁹ −0.59 0.014 −0.16 3.90 ×10⁻⁹ rs10880855 ARID2 T/C 0.51/0.43 JMA 7.83 × 10⁻⁷ 0.790 0.019 0.7794.38 × 10⁻⁸ 0.867 (12: Lower educaton −0.09 1.26 × 10⁻⁷ −0.06 0.067−0.09 8.42 × 10⁻⁹ 46144855) Higher education −0.07 1.60 × 10⁻⁵ −0.160.033 −0.07 3.55 × 10⁻⁶ rs10853531 SLC14A2 G/A 0.70/0.77 JMA 7.82 × 10⁻⁶0.052 0.0023 0.812 2.54 × 10⁻⁸ 0.111 (18: Lower educaton −0.11 1.27 ×10⁻⁶ −0.15 9.01 × −0.11 3.38 × 10⁻⁹ 42824449) 10⁻⁴ Higher education−0.08 2.12 × 10⁻⁶ −0.11 0.288 −0.09 7.14 × 10⁻⁶ JMA, joint meta-analysison SNP association and SNP × education on spherical equivalent; theeffects estimates presented are derived from the JMA for the lower andhigher education group. P_(het), p-value for the test of heterogeneityat each SNP; Allele is listed as risk allele/other allele; FREQ, allelefrequency of the risk allele in Asian/European cohorts.

Example 3 Joint Meta-Analysis in Asians

The JMA for spherical equivalent in 10,315 individuals from the Asianscohorts identified genome-wide significant association for three genes:AREG, GABRR1 and PDE10A (P for JMA<5.0×10⁻⁸; Table 2 and FIG. 3B).SNP×education interaction effects associated with spherical equivalentwere observed at all three loci (P for interaction≤8.48×10⁻⁵). Thegenotype and phenotype associations were highly significant in thehigher education stratum (1.97×10⁻¹⁰≤P≤8.16×10⁻⁸) but were considerablyweaker in the lower education stratum (0.008≤P≤0.243). There was noevidence of inter-study heterogeneity at index SNPs within AREG, GABRR1or PDE10A (Q test: P_(het)≥0.122).

GABRR1 and PDE10A index SNPs were not associated with sphericalequivalent in European samples, for either the JMA joint test, SNP maineffect, or SNP×education interaction (Table 2). AREG SNP rs12511037 wasexcluded in the meta-analysis of European studies after quality controlfiltering (due to MAF<0.05), hence a proxy SNP, rs1246413, in LD withrs12511037 (r²=0.67, D′=1) was tested, whereas insignificant association(P for JMA=0.527; P for interaction=0.176). The meta-regressionincluding study-level characteristics as covariates in the modelconfirmed the heterogeneity between populations of European and Asianancestry (GABRR1 SNP rs13215566: P=0.006; PDE10A SNP rs12206610:P=0.0419). For PDE10A, besides ethnicity, average spherical equivalentof each study also explained the inter-study heterogeneity for theinteraction effects (P=0.025).

It was further examined whether the underlying assumption of G×Eindependence held at these three G×E interaction loci. A meta-analysisof logistic regression analysis was performed for education level onAREG rs12511037, GABRR1 rs13215566 and PDE10A rs12296610, adjusting forage, gender and population stratification in the Singapore cohorts(n=9,004). The analysis did not reveal any significant associationsbetween these loci and education level (P≥0.200, P_(het)≥0.118).Furthermore, the three loci were also not associated with educationalattainment in a large meta-analysis of GWAS recently conducted inEuropean cohorts. Thus, the G×E results are unlikely to be biased due todependence between gene and education.

The association for spherical equivalent in Asian cohorts for four lociidentified from European populations was also evaluated. Two of themwere replicated (FAM150B; P for JMA=0.013; DIS3L-MAP2K1: P forJMA=0.0042; Table 1). DIS3L-MAP2K1 also showed suggestive SNP×educationinteraction in Asians (Pfor interaction=7.95×10⁻⁴), while this was notsignificant in Europeans (P for interaction=0.208).

TABLE 2 Three genetic loci associated with spherical equivalent with asignificant SNP × education interaction in Asian populations, andresults in European populations Asians Europeans (n = 10,315) (n =40,306) SNP (Chr: BP) Gene Allele FREQ Subgroup β P P_(het) β P P_(het)rs12511037 AREG C/T 0.91/0.97 Lower educaiton 0.07 0.243 −0.05 0.323 (4:75334864) Higher education −0.70 1.97 × 10⁻¹⁰ −0.03 0.579 G × E −0.896.87 × 10⁻¹¹ 0.704 0.02 0.176 0.284 JMA 5.55 × 10⁻¹⁰ 0.405 0.527 0.186rs13215566 GABRR1 C/G 0.94/0.84 Lower educaiton −0.13 0.030 −0.03 0.258(6: 89918638) Higher education −0.68 1.46 × 10⁻⁸ −0.01 0.817 G × E −0.568.48 × 10⁻⁵ 0.134 −0.02 0.459 0.457 JMA 3.81 × 10⁻⁸ 0.122 0.502 0.630rs12206610 PDE10A C/T 0.90/0.87 Lower educaiton 0.16 0.008 0.01 0.759 6:166016800 Higher education −0.59 8.16 × 10⁻⁸ 0.01 0.810 G × E −0.72 2.32× 10⁻⁸ 0.920 −0.002 0.421 0.111 JMA 9.21 × 10⁻⁹ 0.902 0.954 0.305 JMA,joint meta-analysis on SNP association and SNP × education on sphericalequivalent; the effects estimates presented are derived from the JMA forthe lower and higher education group. The effect size and p-value forSNP × education interaction were calculated by the meta-analysis ofconducting a 1df Wald test of single interaction parameter (SNP ×education). P_(het), p-value for the test of heterogeneity; Allele islisted as risk allele/other allele; FREQ, allele frequency of the riskallele in Asian/European cohorts. SNP rs12511037 was excluded inmet-analysis in Europeans because of low MAF (MAF < 0.05). Here wepresent the results of a proxy SNP rs1246413 (T/G, FREQ for effectallele T = 0.95) in LD with rs12511037 (r² = 0.67, D′ = 1) forEuropeans.

Example 4 Joint Meta-Analysis of all Cohorts

Subsequently, a combined meta-analysis was conducted, including both theEuropean and Asian subjects of all 34 studies. This analysis revealedtwo additional SNPs: ARID2 (P for JMA=4.38×10⁻⁸) and SLC14A2 (P forJMA=2.54×10⁻⁸). Both loci showed suggestive association with sphericalequivalent in European cohorts, while the association was attenuated inAsian cohorts (Table 1). Genome-wide significant associations were alsodetected with spherical equivalent for 17 known loci identified in theprevious CREAM GWAS.

Example 5 Gene and Education Interactions for GWAS known loci

For the previously reported genetic association with sphericalequivalent at 39 loci identified from recent two large GWAS,interactions with education were evaluated. Two SNP×educationinteractions were nominally significant: TJP2 in Europeans (rs11145488;P for interaction=6.91×10⁻³) and SHISA6-DNAH9 in Asians (rs2969180; Pfor interaction=4.02×10⁻³). In general, the index SNPs tested at 39 locihad larger SNP×education interaction effect on spherical equivalent(meta-regression P for fold changes<0.001). For 20 SNPs with the samedirection of the interaction effect, the magnitudes of interactioneffects were 4-fold larger on average in Asians than in Europeans(meta-regression P=0.003).

Example 6 Gene Expressin in Human Tissues

Using Ocular Tissue Database, the expression of the associated genes wasexamined in 20 normal human donor eyes. The majority of genes identifiedwere expressed in human retina, sclera or retinal pigment epithelium(RPE) (Table 3). Among these genes, GABRR1 had the highest expression inthe retina. The PLIER normalized level of expression of GABRR1 in theretina was 121.7 with an expression value of 21.5 in sclera, suggestingthe expression of GABRR1 is more prevalent within the retina. FAM150Bwas found highly present in the choroid/RPE (expression value of 333.3),while expressed at a much lower level in the retina (29.9). MAP2K1 waswidely expressed in the retina, sclera and choroid with expressionvalues greater than 85.7.

TABLE 3 Gene expression of identified loci in human ocular tissues GENERetina Sclera Choroid/RPE FAM150B 29.94 62.13 333.33 PRL 43.48 24.7443.64 FBN1 12.88 75.26 47.08 MAP2K1 85.72 91.26 183.61 DIS3L 43.20 32.9542.16 SLC14A2 29.96 34.87 33.69 AREG 21.31 26.04 29.64 GABRR1 121.6621.48 31.43 PDE10A 28.19 18.87 21.46 Expression data was obtained fromOcular Tissue Database. The Affymetrix GeneChip Human Exon 1.0 ST (HuEx1.0) microarrays were used to assess gene expression. Normalization ofgene expression was done at both the probe set and metaprobe set levelusing the Probe Logarithmic Intensity Error (PLIER) method withGC-background correction. The PLIER normalized level of gene expressionwas presented in the table.

Discussion:

The above data represent the most comprehensive genome-wide analysis todate of gene and education interactions in relation to refractive error.Novel genetic loci associated with refractive error were identified bytesting the joint contribution of SNP and SNP×education effects in largemulti-ethnic populations. Three genes (AREG, GABRR1 and PDE10A) showedstrong interactions with education in populations of Asian descent.Apart from confirming known associations with spherical equivalent with17 previous published loci, using a combined multiracial cohort, 6 novelloci (FAM150B, LINC00340, FBNI, DIS3L-MAP2K1, ARID2 and SLC14A2)significantly associated with spherical equivalent were identified.

Of the novel loci, GABRR1 on chromosome 6q15 (53 kb) is an especiallyinteresting functional candidate suggestive of a role in myopiadevelopment. Modulation of synaptic plasticity via GABA-mediatedinhibition would be well-placed to alter the “gain” of thevisually-guided feedback system controlling refractive development. Thelead SNP rs12215566 in GABRR1 , together with 7 SNPs within the LD block(r²>0.8), are intronic potentially affecting regulatory motifs (such aszfp128 and gcm1) which may influence transcriptional regulation. As oneof the major inhibitory neurotransmitters in the retina, GABA is activein large retinal cells and amacrine cells. It has been reported thatantagonists to GABA A, B, and C receptors inhibited form-deprivationmyopia in chicks, with greatest effect in the equatorial dimension. GABAreceptors also interact with dopamine pathways in the retina. A recentproteomics study has also showed that levels of GABA transporter-1(GAT-1) are significantly reduced in myopic murine retina after atropinetreatment, implying GABA signaling is involved in anti-myopic effects ofatropine. Therefore, the present result in humans is in line with animalexperiments, supporting the notion that the GABAergic neurotransmittersignaling pathway in the retina could be a potential player in theprogression of myopia.

The rs10889855 on chromosome 6 is an intronic variant within the ARID2gene (AT Rich Interactive Domain 2) and about 500 kb downstream of SNAT1(Solute Carrier Family 38, Member; Aliases SLC38A1). SNAT1 is atransporter of glutamine, a precursor of GABA. It is also highlyexpressed in human retina. In previous meta-analysis in CREAM, variantsin another glutamate receptor gene GRIA4 (encoding glutamate receptor,ionotropic) were identified; altogether current evidence supports thenotion that retinal neurotransmitters GABA and glutamine may be involvedin the refractive development.

The strongest association signal for gene and environment interactionswas from rs12511037, located 14 kb downstream the AREG gene(amphiregulin). AREG is a ligand of the epidermal growth factor receptor(EGFR) promoting the growth of normal epithelial cells, which iscritical for cell differentiation and proliferation such as regrowth ofthe wounded cornea. A link has been found between the muscarinicacetylcholine receptors and the EGFR, as EGFR controls fluid secretionin muscarinic system.

Another novel association, rs16949788 on chromosome 15, derives from aregion that spans DIS3L and MAP2K1. MAP2K1 encodes mitogen-activatedprotein kinase 1 which binds to muscarinic receptors duringproliferation and inhibits the proliferation of human scleralfibroblasts exposed to all-trans retinoic acid. The muscarinicantagonist atropine inhibits the development of myopia in animal modelsand human intervention studies. All-trans retinoic acid is a modulatorof ocular growth, inhibiting the proliferation of human scleralfibroblasts.

FBN1 (Fibrillin 1) encodes a large extracellular matrix glycoprotein, amember of the fibrillin family. Mutations in FBN1 cause Marfan'ssyndrome, a disorder of connective tissue affecting the ocular, skeletaland cardiovascular systems. As a candidate gene for myopia, attempts tostudy its association with myopia previously produced inconclusiveresults, probably due, in part, to underpowered studies withinsufficient sample sizes. Using data from a large multi-ethnicpopulation, the present results support the role of FBN1 in myopiadevelopment.

The genome-wide significant SNPs for the JMA did not exhibit anyinteractions with education in Europeans, in contrast to the significantinteractive effect among Asians. In particular, the interactions ofAREG, GABRR1 and PDE10A with education were evident in Asian populationsonly, but not in Europeans. There are a number of possible reasons.First, the observed heterogeneity may reflect the intense educationsystems in Asia. The higher education level was associated with myopicshift at an average of a 1.16 D in refraction in Asians, but with only a0.56 D in Europeans. It is possible that the gene and educationinterplay may manifest more in such a condition with the strongeducation effects, as genetic effects are generally modest across thepopulations. Second, the population distribution of refractive error ismore myopic in Asians (-0.60 D versus 0.10 D in Europeans). A highprevalence of myopia is likely to associate with other unmeasuredlifestyle exposures, which were not accounted for in the current study.Third, education systems varied widely across studies. We chose todivide education levels into two categories but this cut-off may notreflect the same education intensity or true underlying risk for myopiaacross Europeans and Asians. Any misclassification in environmentmeasurements may bias the effect towards or away from the null. Last,education in adults may not be an accurate surrogate for cumulative nearwork activity. The level of education attained may be a crude marker ofreading intensity and computer use during the crucial years prior to theonset of myopia. These factors, accompanying with varying allelefrequencies at the associated SNPs, might obscure the power to detectthe interaction effects in individuals of European ancestry. Whethersuch G×E interaction is ancestry-specific warrants further evaluation.

Education level reflects the accumulated effect of near work, such asreading and writing. It was thus examined whether there was evidence forSNP×near work interactions associated with spherical equivalent at thethree newly-identified loci (AREG, GABRR1 and PDE10A) in cohorts ofchildren of Asian and European ancestry (combined n=5,835). Tentativesupport for a SNP×near work interaction was observed for PDE10A SNPrs12206610 (Pfor interaction=0.032; P_(het)=0.927). Weaker support foran. interaction was noted at GABRR1 SNP rs13215566 (P forinteraction=0.109; Phet=0.118) and at the AREG SNP rs12511037 (P forinteraction=0.80, Phet=0.224), although the direction of interactioneffect was largely consistent across pediatric studies with thatobserved in adults. A lack of strong SNP×near work associations at theseloci leaves open the possibility that environmental risk exposures otherthan near work might underlie the SNP×education interaction seen in theadult Asian samples.

In summary, 9 novel loci associated with refractive error wereidentified in a large multi-ethnic cohort study by a joint meta-analysisapproach. The present data provide evidence that specific geneticvariants interact with education to influence refractive development,and further support a role for GABA neurotransmitter signaling in myopiadevelopment. These findings provide promising candidate genes forfollow-up work and may lead to new genetic targets for therapeuticinterventions on myopia.

1. A method for the prophylactic or therapeutic treatment of myopia in asubject, comprising administering to the subject an agent that iscapable of decreasing epidermal growth factor receptor (EGFR) signalingand/or signaling of another receptor susceptible for amphiregulin in thesubject in a direct or indirect manner.
 2. The method of claim 1,wherein said agent is selected from the group consisting of: (a) anisolated antibody, antibody fragment or antibody mimetic directedagainst native amphiregulin in said subject; (b) an isolated antibody,antibody fragment or antibody mimetic directed against a nativeamphiregulin precursor in said subject; (c) an isolated antibody,antibody fragment or antibody mimetic directed against EGFR in ablocking manner in said subject; (d) an isolated antibody, antibodyfragment or antibody mimetic directed against another receptorsusceptible for amphiregulin in a blocking manner in a subject; (e) anEGFR inhibiting agent; and (f) a small interfering RNA (siRNA) agentcapable of reducing the expression of amphiregulin in a subject.
 3. Themethod of claim 2, wherein said antibody fragment is selected from thegroup consisting of Fab fragments, F(ab′)₂ fragments and Fab′ fragments.4. The method of claim 2, wherein said antibody mimetic is selected fromthe group consisting of single-chain variable fragments (scFv),single-domain antibodies, affibodies, affilins, affimers, affitins,anticalins, DARPins, monobodies, and peptide aptamers.
 5. The method ofclaim 1, wherein said agent is administered intravitreally,epicorneally, transcorneally, transsclerally, transconjunctivally,subconjunctivally, intraocularly, or systemically.
 6. A method for theprophylactic or therapeutic treatment of hyperopia in a subject,comprising administering to the subject an agent that is capable ofincreasing epidermal growth factor receptor (EGFR) signaling and/orsignaling of another receptor susceptible for amphiregulin in thesubject.
 7. The method of claim 6, wherein said agent is selected fromthe group consisting of: (a) isolated amphiregulin or a biologicallyactive fragment or derivative thereof; (b) an isolated amphiregulinprecursor or a biologically active fragment or derivative thereof; (c)an isolated antibody, antibody fragment or antibody mimetic directedagainst the epidermal growth factor receptor (EGFR) in an activatingmanner in said subject; (d) an isolated antibody, antibody fragment orantibody mimetic directed against another receptor susceptible foramphiregulin in an activating manner in a subject; and (e) an EGFRactivating agent.
 8. The method of claim 7, wherein said amphiregulincomprises the amino acid sequence shown in SEQ ID NO:
 1. 9. The methodof claim 7, wherein said amphiregulin consists of the amino acidsequence shown in SEQ ID NO:
 1. 10. The method of claim 7, wherein saidbiologically active fragment or derivative of amphiregulin comprises anamino acid sequence having at least 50% sequence identity to the aminoacid sequence shown in SEQ ID NO:
 1. 11. The method of claim 7, whereinsaid antibody fragment is selected from the group consisting of Fabfragments, F(ab′)₂ fragments and Fab′ fragments.
 12. The method of claim7, wherein said antibody mimetic is selected from the group consistingof single-chain variable fragments (scFv), single-domain antibodies,affibodies, affilins, affimers, affitins, anticalins, DARPins,monobodies, and peptide aptamers.
 13. The method of claim 6, whereinsaid agent is administered intravitreally, epicorneally, transcorneally,transsclerally, transconjunctivally, subconjunctivally, intraocularly,or systemically.
 14. A method for the diagnosis of myopia or hyperopia,or of a predisposition for the development of myopia or hyperopia, in asubject, comprising the steps of: (a) providing a biological sample fromthe subject; (b) determining the amphiregulin level in said sample; (c)comparing the level determined in step (b) to the amphiregulin levelsfound in emmetropic subjects or subjects going to be emmetropic; and (d)determining that the subject has myopia or is predisposed for thedevelopment of myopia in case the level determined in step (b) is higherthan the amphiregulin levels found in emmetropic subjects or subjectsgoing to be emmetropic; and determining that the subject has hyperopiaor is predisposed for the development of hyperopia in case the leveldetermined in step (b) is lower than the amphiregulin levels found inemmetropic subjects or subjects going to be emmetropic.
 15. A method foridentifying agents which associate with amphiregulin or fragments orvariants thereof, comprising the steps of: (a) contacting saidamphiregulin or fragments or variants thereof, or a recombinant hostcell expressing amphiregulin or fragments or variants thereof, with atest compound and (b) analyzing the ability of said test compound tobind to amphiregulin or fragments or variants thereof.